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The GNAQ T96S Mutation Affects Cell Signaling and Enhances the Oncogenic Properties of Hepatocellular Carcinoma

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International Journal of Molecular Sciences Article The GNAQ T96S Mutation Affects Cell Signaling and Enhances the Oncogenic Properties of Hepatocellular Carcinoma Eugene Choi 1, Sung Jean Park 2, Gunhee Lee 3, Seung Kew Yoon 4 , Minho Lee 5 and Suk Kyeong Lee 1,* 1 Department of Medical Life Sciences, Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea; [email protected] 2 College of Pharmacy and Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon 21936, Korea; [email protected] 3 Precision Medicine Research Center, Integrated Research Center for Genome Polymorphism, Department of Microbiology, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea; [email protected] 4 Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea; [email protected] 5 Department of Life Science, Dongguk University-Seoul, Ilsandong-gu, Goyang-si 10326, Gyeonggi-do, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-2-2258-7480 Abstract: Hepatocellular carcinoma (HCC), the most common malignant tumor in the liver, grows and metastasizes rapidly. Despite advances in treatment modalities, the five-year survival rate of HCC remains less than 30%. We sought genetic mutations that may affect the oncogenic properties of HCC, using The Cancer Genome Atlas (TCGA) data analysis. We found that the GNAQ T96S mutation (threonine 96 to serine alteration of the Gαq protein) was present in 12 out of 373 HCC patients (3.2%). To examine the effect of the GNAQ T96S mutation on HCC, we transfected the Citation: Choi, E.; Park, S.J.; Lee, G.; SK-Hep-1 cell line with the wild-type or the mutant GNAQ T96S expression vector. Transfection Yoon, S.K.; Lee, M.; Lee, S.K. The with the wild-type GNAQ expression vector enhanced anchorage-independent growth, migration, GNAQ T96S Mutation Affects Cell and the MAPK pathways in the SK-Hep-1 cells compared to control vector transfection. Moreover, Signaling and Enhances the Oncogenic Properties of cell proliferation, anchorage-independent growth, migration, and the MAPK pathways were further Hepatocellular Carcinoma. Int. J. Mol. enhanced in the SK-Hep-1 cells transfected with the GNAQ T96S expression vector compared to the Sci. 2021, 22, 3284. https://doi.org/ wild-type GNAQ-transfected cells. In silico structural analysis shows that the substitution of the 10.3390/ijms22063284 GNAQ amino acid threonine 96 with a serine may destabilize the interaction between the regulator of G protein signaling (RGS) protein and GNAQ. This may reduce the inhibitory effect of RGS on Academic Editor: Paola Ghiorzo GNAQ signaling, enhancing the GNAQ signaling pathway. Single nucleotide polymorphism (SNP) genotyping analysis for Korean HCC patients shows that the GNAQ T96S mutation was found in Received: 5 March 2021 only one of the 456 patients (0.22%). Our data suggest that the GNAQ T96S hotspot mutation may Accepted: 20 March 2021 play an oncogenic role in HCC by potentiating the GNAQ signal transduction pathway. Published: 23 March 2021 Keywords: hepatocellular carcinoma; guanine nucleotide-binding protein G(q) subunit alpha; helical Publisher’s Note: MDPI stays neutral domain; somatic mutation; mitogen-activated protein kinase (MAPK) signaling with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Liver cancer is globally the sixth-most-common type of cancer and the fourth-most- common cause of cancer mortality [1]. Single nucleotide polymorphisms (SNPs) in liver Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. cancer have been studied as a cancer risk factor contributing to poor prognosis in pa- This article is an open access article tients [2–4]. The somatic mutation S1113R of the phosphatidylinositol-3,4,5-trisphosphate distributed under the terms and dependent Rac exchange factor 2 (PREX2) gene has been shown to promote migration conditions of the Creative Commons and proliferation and activate the AKT pathway in hepatocellular carcinoma (HCC) [5]. Attribution (CC BY) license (https:// Meanwhile, the polymorphisms of UDP-glucuronosyltransferase 1A enzymes contribute to creativecommons.org/licenses/by/ HCC development in vivo [6], suggesting the potential of single amino acid substitutions 4.0/). as a biomarker for liver cancer. Int. J. Mol. Sci. 2021, 22, 3284. https://doi.org/10.3390/ijms22063284 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 3284 2 of 14 The G protein plays an essential role in cellular signal transduction. The somatic mutation R201H in gnas (encoding Gαs) promotes pancreatic tumorigenesis in murine models that recapitulates human intraductal papillary mucinous neoplasm [7]. Moreover, the Arg-200 mutation in GNA13 (encoding Gα13) has been proposed as a putative driver of bladder cancer [8]. Guanine nucleotide-binding protein G(q) subunit alpha (GNAQ, encoding Gαq), GNAS, and GNA14 (encoding Gα14) mutations were also found in hepatic small vessel neoplasm and intrahepatic cholangiocarcinoma in the liver, although their roles have not been elucidated [9,10]. Thus, functional studies on the effects of G protein mutations in liver cancer are needed. Gαq is a component of a heterotrimeric protein that binds with guanosine triphosphate (GTP) and cycles between active and inactive states by hydrolyzing GTP to guanosine diphosphate (GDP). The Gαq protein consists of 359 amino acids and contains two primary domains: the GTPase domain and the helical domain. Within the GTPase domain are three switch regions [11], which are the primary contact sites for the Gβγ subunit (Switch II), the regulator of G protein signaling (RGS), and the effector. The helical domain consists entirely of α helices and is characterized by a unique sequence for each Gα protein [12]. Gαq is one of the most critical modulators and transducers of various transmembrane signaling systems. It mediates many signaling pathways, including the mitogen-activated protein kinase (MAPK) signaling pathway [13–15]. When the whole genome sequence data for 11,119 cancer tissues of 41 cancer types were analyzed, common recurrent hotspot mutations of several genes were identified in various cancers [16]. Among the recurrent mutations in GNAQ, new hotspot mutations were identified at codons 96 and 48, in addition to the already-known mutations at codons 183 and 209. The codon 96 mutation is expressed in the helical domain of Gαq and is a missense mutation in which nucleotide 286 adenine is changed to thymine, resulting in amino acid 96 changing from threonine to serine (hereafter referred to as “T96S”) [16]. The GNAQ T96S mutation has been reported in various diseases. In gastric cancer (GC), the GNAQ T96S mutation was predicted to bind with the highly frequent HLA allele as a tumor-specific neoantigen, and suggested as a potential target for T cell immunother- apy [17]. GNAQ T96S was observed as a rare and frequent mutation in prostate cancer and non-small-cell lung cancer (NSCLC), respectively [13,18]. The GNAQ T96S mutation was also detected in a three-year-old boy with diffuse bone and soft tissue angiomatosis [19]. Besides, In natural killer/T cell lymphoma (NKTCL), the GNAQ T96S mutation shows strong dominant-negative effects in both in vitro and in vivo experiments [14]. However, few studies report the role of GNAQ T96S in HCC. We investigated the role of the GNAQ T96S mutation in HCC by analyzing the effect of GNAQ T96S overexpression on various phenotypes and intracellular signaling pathways in HCC cells. 2. Results 2.1. The Cancer Genome Atlas (TCGA) Genotype Data in Liver Cancer Patients with HCC We investigated the liver hepatocellular carcinoma (LIHC) category in the Broad Institute’s Genome Data Analysis Center (GDAC; https://gdac.broadinstitute.org, accessed date: 4 September 2018). We found the GNAQ T96S somatic mutation in samples of 12 patients out of 373 HCC patients (3.2%). All patients with the mutation were male and had a heterozygous genotype on the locus. Four of the 12 patients were White; eight were Asian. The age range was quite wide (18–80, average 54.83, Supplementary Data S1). We also investigated whether GNAQ T96S was associated with other liver cancer risk factors, including sex, alcohol consumption, alpha-1 antitrypsin deficiency, hemochromato- sis, hepatitis B, hepatitis C, and nonalcoholic fatty liver disease. We found no association with other liver cancer risk factors except sex (Table1, Supplementary data S2). Int. J. Mol. Sci. 2021, 22, 3284 3 of 14 Table 1. Information on samples and patients having the guanine nucleotide-binding protein G(q) subunit alpha (GNAQ) T96S mutation in TCGA. Tumor Sample Matched Normal Race Age Sex CC-A8HS-01A-11D-A35Z-10 CC-A8HS-10A-01D-A35Z-10 Asian 18 M DD-A4NF-01A-11D-A27I-10 DD-A4NF-10A-01D-A27I-10 White 72 M DD-AACW-01A-11D-A40R-10 DD-AACW-10A-01D-A40U-10 Asian 43 M DD-AADG-01A-11D-A40R-10 DD-AADG-10A-01D-A40U-10 Asian 70 M DD-AADV-01A-11D-A38X-10 DD-AADV-10A-01D-A38X-10 Asian 50 M DD-AADW-01A-11D-A38X-10 DD-AADW-10A-01D-A38X-10 Asian 48 M DD-AAE8-01A-11D-A40R-10 DD-AAE8-10A-01D-A40U-10 Asian 45 M DD-AAEK-01A-11D-A40R-10 DD-AAEK-10A-01D-A40U-10 Asian 51 M ED-A97K-01A-21D-A382-10 ED-A97K-10A-01D-A385-10 Asian 54 M G3-A3CG-01A-11D-A20W-10 G3-A3CG-10A-01D-A20W-10 White 80 M K7-A5RF-01A-11D-A28X-10 K7-A5RF-10B-01D-A28X-10 White 64 M MI-A75G-01A-11D-A32G-10 MI-A75G-10A-01D-A32G-10 White 63 M 2.2. Sequencing and Overexpression of the GNAQ T96S Hotspot Mutation in SK-Hep-1 Cells Hotspot mutations located in GNAQ are marked in a schematic diagram of GNAQ (Figure1A).
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  • Supporting Information

    Supporting Information

    Supporting Information Jin et al. 10.1073/pnas.1418629112 SI Materials and Methods Santa Cruz Biotechnology, 1:100), anti–neural-specific β-tubulin − − − − Animals. Mice carrying the Gas1 (1), Shh (2), Cdo , Boc (3), (Tuj1, mouse, Millipore, 1:800), anti-Neurotrophin receptor P75 f − Smo (4), Gnaz (5), and transgenic Wnt1:Cre (6) alleles were (Rabbit, Millipore, 1:200), anti-GFP (rabbit, Invitrogen, 1:100), − − − previously described. For simplicity, Gas1 , Cdo ,andBoc were anti-mouse Gas1 (goat, R&D Systems, 1:200), and anti-HuC/D used for the Lac-Z knock-in allele of Gas1 and β-geo–internal (mouse, Molecular Probes, 1:100). Alexa Fluor 488- and Alexa ribosomal entry site (IRES) human placental alkaline phosphatase Fluor 568-conjugated secondary antibodies (Invitrogen) against (hPLAP) gene-trapped alleles of Cdo and Boc, respectively. Ap- specific species (goat, mouse, and rabbit) were used for detection propriate mating schemes were designed to generate embryos of (Molecular Probes, all at 1:1,000). DAPI (Sigma) was used at desired genotypes, including controls. Embryo stages are specified 1 μg/mL for staining of DNA. in the text. The vaginal plug date is designated as embryonic day 0.5 (E0.5), following convention. For genotyping, tail or embryo Neurosphere Culture. Neurosphere-like bodies were generated −/− sac DNAs were used. Oligonucleotide primers and conditions using whole guts dissected from E11.5 wild-type or Gas1 for PCR are described in corresponding publications and on the embryos as previously described (7–9). They were dissociated in f Jackson Laboratory (JAX) website. The Gas1 allele was gener- basal media by mechanical pipetting, then plated into culture ated for this work, and its characterization is detailed in Fig.